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The three phosphate groups of theATP, shown in yellow, are visible. In this case,bases in both the anticodon arm and the aminoacid arm are the key structural features of thetRNA used for recognition by the aminoacyl-tRNAsynthetase. Additional contacts between the enzymeand the tRNA revealed in this crystal structureoccur along the inside of the L structure of thetRNA (see Fig. 26-17), but many of these involveresidues conserved in all tRNAs and may not contribute to discrimination among different tRNAs.Direction of chain growthAmino terminusCarboxyl terminusA complete understanding of the structural factors guiding theseinteractions remains an area of very active investigation.
The solutionof the crystal structures of two aminoacyl-tRNA synthetases (Gin andAsp) complexed with their cognate tRNAs and ATP is an importantadvance (Fig. 26-22). The relatively simple alanine system describedabove may be an evolutionary relic of a period when RNA oligonucleotides (ancestors to tRNA) were aminoacylated in a primitive system forprotein synthesis.Polypeptide Synthesis Begins at the Amino-Terminal End146Residue numberFigure 26-23 Proof that polypeptide chains growby addition of new amino acid residues to the carboxyl end. The dark red zones show the portions ofcompleted a-globin chains containing radioactiveLeu residues at different times after addition oflabeled leucine.
At 4 min, only a few residues atthe carboxyl end of a-globin were labeled. This isbecause the only complete globin chains that contained label after 4 min were those that had nearlycompleted synthesis at the time the label wasadded. On longer times of incubation with labeledleucine, successively longer segments of the polypeptide chain contained labeled residues, always ina block at the carboxyl end of the chain. The unlabeled end of the polypeptide (the amino terminus)was thus defined as the initiating end, and thepolypeptide chain grows by successive addition ofamino acids at the carboxyl end.Does polypeptide chain growth begin from the amino-terminal or fromthe carboxyl-terminal end? The answer came from isotope tracer experiments carried out by Howard Dintzis in 1961.
Reticulocytes (immature erythrocytes) that were actively synthesizing hemoglobin wereincubated with radioactive leucine. Leucine was chosen because it occurs frequently along both the a- and /3-globin chains. Samples of completed a chains were isolated from the reticulocytes at various timesafter addition of radioactive leucine, and the distribution of radioactivity along the a chain was determined with the expectation that itwould be concentrated in the end that was synthesized last.
In thoseglobin chains isolated after 60 min of incubation, nearly all the Leuresidues were radioactive. However, in completed globin chains thatwere isolated only a few minutes after radioactive leucine was added,radioactive Leu residues were concentrated at the carboxyl-terminalend (Fig. 26-23). From these observations it was concluded that polypeptide chains are begun at the amino-terminal end and are elongatedby sequential addition of residues to the carboxyl-terminal end. Thispattern has been confirmed in innumerable additional experimentsand applies to all proteins in all cells.A Specific Amino Acid Initiates Protein SynthesisHCOO"I IH—C—N—C—HOCH 2CH 2SA^-Formylmethionme916ICH 3Although there is only one codon for methionine (AUG), there are twotRNAs for methionine in all organisms.
One tRNA is used exclusivelywhen AUG represents the initiation codon for protein synthesis. Thesecond is used when methionine is added at an internal position in apolypeptide.In bacteria, the two separate classes of tRNA specific for methionine are designated tRNAMet and tRNA™61. The starting amino acidresidue at the amino-terminal end is iV-formylmethionine. It entersChapter 26 Protein Metabolism917the ribosome as iV-formylmethionyl-tRNA™61 (f Met-tRNA™^), whichis formed in two successive reactions. First, methionine is attached totRNA™61 by the Met-tRNA synthetase:Methionine + tRNA™61 + ATPMet-tRNA™* + AMP + PP;As already noted, there is only one of theseaminoacylates both tRNAfMet and tRNAMet.transferred to the amino group of theformyltetrahydrofolate by a transformylaseenzymes in E.
coli, and itSecond, a formyl group isMet residue from N10enzyme:iV^-Formyltetrahydrofolate + Met-tRNA™^>tetrahydrofolate + fMet-tRNA™61This transformylase is more selective than the Met-tRNA synthetase,and it cannot formylate free methionine or Met residues attached totRNAMet. Instead, it is specific for Met residues attached to tRNA™61,presumably recognizing some unique structural feature of that tRNA.The other Met-tRNA species, Met-tRNAMet, is used to insert methionine in interior positions in the polypeptide chain.
Blocking of theamino group of methionine by the AT-formyl group not only prevents itfrom entering interior positions but also allows fMet-tRNAfMet to bebound at a specific initiation site on the ribosome that does not acceptMet-tRNAMet or any other aminoacyl-tRNA.In eukaryotic cells, all polypeptides synthesized by cytosolic ribosomes begin with a Met residue (as opposed to f Met), but again a specialized initiating tRNA is used that is distinct from the tRNAMet usedat interior positions. In contrast, polypeptides synthesized by the ribosomes in the mitochondria and chloroplasts of eukaryotic cells beginwith Af-formylmethionine. This and other similarities in the proteinsynthesizing machinery of these organelles and bacteria strongly support the view that mitochondria and chloroplasts originated from bacterial ancestors symbiotically incorporated into the precursors of eukaryotic cells at an early stage of evolution (see Fig.
2-17).We are now left with a puzzle. There is only one codon for methionine, namely (5')AUG. How can this single codon serve to identify boththe starting A^-formylmethionine (or methionine in the case of eukaryotes) and those Met residues that occur in interior positions in polypeptide chains? The answer will be found in the next section.30SSubunitInitiation of Polypeptide Synthesis Has Several StepsWe now turn to a detailed examination of the second stage of proteinsynthesis: initiation.
The focus here, and in the discussion of elongation and termination to follow, is on protein synthesis in bacteria; theprocess is not as well understood in eukaryotes. The initiation of polypeptide synthesis in bacteria requires (1) the 30S ribosomal subunit,which contains 16S rRNA, (2) the mRNA coding for the polypeptide tobe made, (3) the initiating f Met-tRNA™61, (4) a set of three proteinscalled initiation factors (IF-1, IF-2, and IF-3), (5) GTP, (6) the 50S ribosomal subunit, and (7) Mg 2+ .
The formation of the initiation complextakes place in three steps (Fig. 26-24).In the first step, the 30S ribosomal subunit binds initiation factor 3(IF-3), which prevents the 30S and 50S subunits from combining prematurely. Binding of the mRNA to the 30S subunit then takes place insuch a way that the initiation codon (AUG) binds to a precise locationon the 30S subunit (Fig. 26-24).5'NextcodonFigure 26-24 Formation of the initiation complexin three steps (described in the text) at the expenseof the hydrolysis of GTP to GDP and Pj. IF-2 andIF-3 are initiation factors. P designates the peptidylsite, A the aminoacyl site.918Part IV Information PathwaysE.colitrpA (5') A G C A CE.coliaraBU U U G G A UE.colilacl<£X174 phage A proteinAphagecroGA A AU G AG IC U UC A A U U C A GA A U C U UA U G U A CU C U G A U G G A A C G C U AA A C G A U GG C G A U U G CU G A A U G U GA A A C C A G UU U U U A U GG U U C G U U CU G U A U GG A A C A A C GShine-Dalgarno sequence;pairs with 16S rRNAC (3')AAUCInitiation codon;pairs with fMet-tRNA™^(a)3'OH3' End of16S rRNAiGAUAUU C C U C C AcIdealizedprokaryoticmRNA(5')G A U U C C IUUGACC U A U G C G A G C U U U U A G U(3')(b)Figure 26-25 Sequences on the mRNA that serveas signals for initiation of protein synthesis in prokaryotes.
(a) Alignment of the initiating AUG(shaded in green) in the P site depends in part onShine-Dalgarno sequences (shaded in red) upstream. Portions of the mRNA transcripts of fiveprokaryotic genes are shown, (b) The ShineDalgarno sequences pair with a sequence near the3' end of the 16S rRNA, as shown for an idealizedprokaryotic mRNA.The initiating AUG is guided to the correct position on the 30Ssubunit by an initiating signal called the Shine-Dalgarno sequencein the mRNA, centered 8 to 13 base pairs to the 5' side of the initiationcodon (Fig.
26-25). Generally consisting of four to nine purine residues, the Shine-Dalgarno sequence is recognized by, and base-pairs(antiparallel) with, a complementary pyrimidine-rich sequence nearthe 3' end of the 16S rRNA of the 30S subunit. This mRNA-rRNAinteraction fixes the mRNA so that the AUG is correctly positioned forinitiation of translation. The specific AUG where f Met-tRNA™6* is tobe bound is thereby distinguished from interior methionine codons byits proximity to the Shine-Dalgarno sequence in the mRNA.Ribosomes have two sites that bind aminoacyl-tRNAs, the aminoacyl or A site and the peptidyl or P site.
Both the 30S and the 50Ssubunits contribute to the characteristics of each site. The initiatingAUG is positioned in the P site, which is the only site to which fMettRNA™61 can bind (Fig. 26-24). However, f Met-tRNA™* is the exception: during the subsequent elongation stage, all other incoming aminoacyl-tRNAs, including the Met-tRNAMet that binds to interior AUGs,bind to the A site.
The P site is the site from which the "uncharged"tRNAs leave during elongation.In the second step of the initiation process (Fig. 26-24), the complex consisting of the 30S subunit, IF-3, and mRNA now forms a stilllarger complex by binding IF-2, which already is bound to GTP and theinitiating f Met-tRNA™^. The anticodon of this tRNA pairs correctlywith the initiation codon in this step.In the third step, this large complex combines with the 50S ribosomal subunit; simultaneously, the GTP molecule bound to IF-2 is hydrolyzed to GDP and Pi (which are released).
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